The broad objective of the proposed research is to determine the biological function of alpha-actinin in biological movement systems and how this function is related to the physiological roles of actin and actin-binding proteins such as tropomyosin, filamin, profilin, and others in movement. Actin seems to have both cytoskeletal and cytocontractile functions in cells, and alpha-actinin has been proposed to bind actin filaments to each other and to plasma membranes in a variety of cells. Attempts will be made to detect and locate alpha-actinin and other actin-binding proteins that may function synergistically or antagonistically with alpha-actinin in different nonmuscle cells; these attempts will use antibody localization techniques and biochemical assays of cells. Studies will be done of alpha-actinin-actin and alpha-actinin-membrane interactions in the presence of actin and different actin-binding proteins; these studies will involve in vitro assays using purified proteins and specific proteolysis with a Ca2 ion-activated protease. Subsequently, attempts will be made to determine whether any interaction, possibly mediated by alpha-actinin or an actin-binding protein, exists among the three filament systems found in most animal cells: 1) the microfilament system; 2) 10-nm intermediate filaments; and 3) microtubules. These efforts will use mixtures of purified proteins such as actin, alpha-actinin, tubulin, and desmin and various measurements to detect binding in these mixtures. Attempts will also be made to learn the effects of microinjected alpha-actinin antibodies or alpha-actinin on microfilament organization in cultured cells; these studies will use phase and fluorescence microscopy of cultured cells after microinjection with fluorescently labeled, biologically active molecules.